This invention relates generally to methods for obtaining bone precursor cells and compositions comprising such cells. The invention includes methods for enriching the population of bone precursor cells in bone marrow cells isolated from mammalian bones or peripheral blood. Also provided are methods for differentiating bone precursor cells into osteoblasts, and diagnostic and even prognostic methods.
The rate of bone fractures in the United States is estimated at 6,000,000 individuals per year. In 1984 (Holbrock et al., 1984) these injuries resulted in a direct cost (i.e., excluding loss of income) of $17,000,000,000 per year. When a bone is completely fractured, a significant number of fractures require medical intervention beyond simple immobilization (casting), particularly those involving trauma. A major problem in such instances is the lack of proximity of the two bone ends (referred to as non-union). This results in an inappropriate and prolonged repair process, which may prevent recovery.
The average length of time for the body to repair a fracture is 25-100 days, for moderate load-bearing, and one year for complete repair. Thus, both simple fractures and medically complicated breaks would benefit from novel therapeutic modalities which accelerate and/or complete the repair process. The same is true for those bone diseases (referred to as osteopenias) which result in a thinning of the bone the primary symptom of which is an often-debilitating fracture.
Primary Osteoporosis is an increased, progressive bone loss which accompanies the aging process. As such, it represents significant health risk in the United States which greater than 15 million Americans suffering from primary (idiopathic) osteoporosis resulting in a direct cost of $6,000,000,000 per year (Holbrock et al., 1984). Primary osteoporosis is the most common of the metabolic bone diseases, and some 40,000-50,000 fracture-related deaths per year are attributed to this disorder. This mortality rate is greater than deaths due to cancer of the breast and uterus, combined. Significantly, this disorder, which is one of the osteopenias, is asymptomatic until a bone fracture occurs. Affected individuals typically fracture the radius, femoral head, or collapse vertebrae.
Osteoporosis has a greater impact on the female population with larger numbers of women than men struck by this disorder, and a significant increase in the rate of osteoporosis occurs post-menopause. The rate of osteoporosis in these women is slowed but not ameliorated by estrogen replacement therapy. Indeed, there is no convincing medical evidence that any treatment is successful in restoring lost bone mass of any kind. Given the aging of the American population, patients with osteoporosis also represent a significant target population for effective and novel bone therapies.
The process of aging in general is associated with a progressive diminution of bone-accumulation capacity, especially in trabecular bone (Nimni et al., 1993). This decreased structural integrity is associated with a number of alterations in bone proteins, osteoid formation, calcium loss etc., leading to osteopenia (Nimni et al., 1993; Fedarko et al., 1992; Termine, 1990). The exact cellular mechanisms underlying such changes in bone structure and function are unclear. However, central to all of these alterations are cells of the osteoblast lineage.
Reductions in osteoblast function or numbers, of necessity, leads to the loss of bone-forming capacity. It is known that some aspects of osteoblast function decrease greatly with age (Termine, 1990). Overall, total protein synthesis and the synthesis of specific proteoglycans decreases markedly (Fedarko et al., 1992), whereas collagen and other proteins such as fibronectin and thrombospondin are degraded (Termine, 1990).
Bone cells from older individuals, in vitro, have the capacity to respond to growth factors, but their synthetic and proliferative capacity is diminished (Termine, 1990), presumably due to reduced responsiveness to various osteogenic growth factors (Pfeilschifter et al., 1993). This results in diminished bone precursor cell and osteoblast numbers (Nimni et al., 1993).
There is no current treatment for lost bone mass, including various growth-promoting proteins and Vitamin D3. Likewise, there is no effective replacement or implant for non-union fractures or crush injuries of the bone. Currently, these latter types of injury utilize bovine (cow), or human cadaver bone which is chemically treated (to remove proteins) in order to prevent rejection. However, such bone implants, while mechanically important, are biologically dead (they do not contain bone-forming cells, growth factors, or other regulatory proteins). Thus, they do not greatly modulate the repair process. All of these concerns demonstrate a great need for new or novel forms of bone therapy.
Bone development results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells, and the eventual calcification of cartilage and bone extracellular matrix (Urist et al., 1983). Human bone marrow contains a distinct cell population that expresses bone proteins and responds to growth factor xcex21 (TGF-xcex2), but not to hematopoietic growth factors (Long et al., 1990).
Little information exists concerning the growth factors or cytokines controlling development of bone precursor cells (osteoprogenitor cells and preosteoblasts) into their differentiated progeny, the osteoblasts. Likewise, few studies address the impact of extracellular matrix (ECM) molecules on this stage of human bone cell development, or the impact of aging on either of these two areas. In the past, human bone cells (both precursor cells and osteoblasts) have been technically difficult to acquire and purification/characterization studies or protocols were few in number. Additionally, current in vitro models of bone formation are limited as the use of post-fetal mesenchymal tissue to generate bone cells often results in chondrogenesis, but is inadequate for osteogenesis, (Urist et al., 1983). Thus, information concerning the cellular activation signals, differentiation, and bone matrix production during the early phases of human bone cell development is limited, at best.
The regulation of chondro-osteogenic gene activation is induced during bone morphogenesis by an accumulation of extracellular and intracellular signals (Urist et al., 1983). Importantly, extracellular signals are known to be transferred from both cytokines and extracellular matrix molecules (Urist et al., 1983), to responding cell surface receptor(s) resulting in eventual bone formation. The formation of bone occurs by two mechanisms. Direct development of bone from mesenchymal cells (referred to as intramembranous ossification; as observed in skull formation) occurs when mesenchymal cells directly differentiate into bone tissue. The second type of bone formation (the endochondral bone formation of skeletal bone) occurs via an intervening cartilage model.
The development and growth of long bones thus results from the proliferation of mesenchymal cells, their differentiation into osteogenic progenitor cells and (then) osteoblasts, cartilage deposition, and eventual calcification of the cartilage and/or bone matrix. Concurrently, bone is remodeled to form a tubular bone space in which hematopoietic cell differentiation occurs.
Interestingly, the number of osteoprogenitor cells in adult bone seems too small to replace all of the large mass of bone normally remodeled in the process of aging of skeleton (Urist et al., 1983). Further observations (vide infra) confirm this concept by showing that one (unexpected) source of osteoprogenitor cells is the bone marrow. This reduced progenitor cell number also implies that there is a disassociation of bone progenitor cell recruitment from subsequent osteogenic activation and bone deposition, and further suggests multiple levels of regulation in this process.
One of the central issues concerning bone formation regards the developmental lineages of the bone cell types, namely the osteoblast and the osteoclast. There is adequate evidence to suggest that osteoblasts arise from local mesenchymal cell populations, and that osteoclasts are derived from blood-born monocyte/macrophage cells.
Fischman and Haye first demonstrated that monocytes fused to form osteoclasts in regenerating newt limbs (Fishman et al., 1962). Although the role of macrophage fusion remains controversial (Hattersley et al., 1989 and Horton et al., 1985), further evidence for the blood-born origin of the osteoclast was pioneered by LeDouarin using a chick:quail chimera in which nuclear morphology allows clear distinction of cell derivation. These studies conclusively demonstrated that osteoblasts and osteocytes are derived from the limb bud mesenchyma whereas osteoclasts arise from blood-born hematopoietic cells (Jotereau et al., 1978 and Le Douarin, 1973). The importance of these observations was subsequently shown by the successful cure (by osteoclasts) of osteopetrosis utilizing bone marrow transplantation in both animals (Ash et al., 1980), and humans (Coccia et al., 1980). While such data conclusively show the hematogenous origin of the osteoclast, little knowledge exists on the nature or location of the stem cell population(s) capable of differentiating into bone-forming osteoblasts.
Like other developing tissues, bone responds to bone-specific, and other soluble growth factors. TGF-xcex2 is a member of a family of polypeptide growth regulators that affect cell growth and differentiation during developmental processes, such as embryogenesis and tissue repair (Sporn et al., 1985). TGF-xcex2 strongly inhibits proliferation of normal and tumor-derived epithelial cells, blocks adipogenesis, myogenesis, and hematopoiesis (Sporn et al., 1985). However, in bone, TGF-xcex2 is a positive regulator.
TGF-xcex2 is localized in active centers of bone differentiation (cartilage canals and osteocytes) (Massague, 1987), and TGF-xcex2 is found in high quantity in bonexe2x80x94suggesting that bone contains the greatest total amount of TGF-xcex2 (Massague, 1987 and Gehron Robey et al., 1987). During bone formation, TGF-xcex2 also promotes chondrogenesis (Massague, 1987)xe2x80x94an effect presumably related to its ability to stimulate the deposition of extracellular matrix (ECM) components (Ignotz et al., 1986). Besides stimulating cartilage formation, TGF-xcex2 is synthesized and secreted in bone cell cultures, and stimulates the growth of sub-confluent layers of fetal bovine bone cells, thus showing it to be an autocrine regulator of bone cell development (Sporn et al., 1985).
In addition to TGF-xcex2, other growth factors or cytokines are implicated in bone development. Urist and co-workers have been able to isolate various regulatory proteins that function in both in vivo and in vitro models (Urist et al., 1983). Bone morphogenic protein (BMP), originally an extract of demineralized human bone matrix, has now been cloned (Wozney et al., 1988), and when implanted in vivo results in a sequence of events leading to functional bone formation (Wozney et al., 1988 and Muthukumaran et al., 1985). The implanting of BMP is followed by mesenchymal cell migration to the area of the implant, differentiation into bone progenitor cells, deposition of new bone, and subsequent bone remodeling to allow the establishment of bone marrow (Muthukumaran et al., 1985).
A number of additional growth factors exist which regulate bone development. In particular, bone-derived growth factors (BDGF) stimulate bone cells to proliferate in serum-free media (Hanamura et al., 1980 and Linkhart et al., 1986). However, these factors seem to function at a different level from BMP (Urist et al., 1983).
The extracellular matrix (ECM) varies in its tissue composition throughout the body, consisting of various components such as collagen, proteoglycan, and glycoprotein (Wicha et al., 1982). Numerous studies point to the influences of ECM in promoting cellular development. Gospodarowicz et al. demonstrated that ECM, the natural substrate surrounding cells in vivo, greatly affects corneal epithelial cell proliferation in vitro (Gospodarowicz and Ill, 1980 and Gospodarowicz et al., 1980). Studies by Reh et al. (1987) show that extracellular components such as laminin are involved in inductive interactions which give rise to retinal and retinal pigmented endothelium. Also, differentiation and growth of mammary epithelial cells are profoundly influenced by ECM components, and mammary cell growth in vivo and in vitro requires type IV collagen (Wicha et al., 1982), Finally, studies from one of the inventor""s laboratories show that bone marrow ECM also plays a major role in hematopoiesis in that complex ECM extracts greatly augment cell proliferation (Campbell et al., 1985), and that marrow-derived ECM contains specific cytoadhesion molecules (Campbell et al., 1987; Campbell et al., 1990; Long and Dixit, 1990; Long et al., 1990; and Long et al., 1992).
A number of non-collagenous matrix proteins, isolated from demineralized bone, are involved in bone formation. Osteonectin is a 32 kDa protein which, binding to calcium, hydroxyapatite and collagen, is believed to initiate nucleation during the mineral phase of bone deposition (Termine et al., 1981). In vivo analysis of osteonectin message reveals its presence in a variety of developing tissues (Nomura et al., 1988 and Holland et al., 1987). However, it is present in its highest levels in bones of the axial skeleton, skull, and the blood platelet (megakaryocyte) (Nomura et al., 1988).
Bone gla-protein (BGP, osteocalcin) is a vitamin K-dependent, 5700 Da calcium binding bone protein that is specific for bone and may regulate Ca++ deposition (Termine et al., 1981; Price et al., 1976; and Price et al., 1981). Other bone proteins seem to function as cytoadhesion molecules (Oldberg et al., 1981 and Somerman et al., 1987), or have unresolved functions (Reddi, 1981).
While bone morphogenesis is ECM dependent, bone ECM also contains a number of the more common mesenchymal growth factors such as PDGF, basic, and acidic fibroblast growth factor (Urist et al., 1983; Linkhart et al., 1986; Hauschka et al., 1986; and Canalis et al., 1985). These activities are capable of stimulating the proliferation of mesenchymal target cells (BALB/c 3T3 fibroblasts, capillary endothelial cells, and rat fetal osteoblasts). As well, bone-specific proliferating activities such as the BMP exist in bone ECM.
While these general and specific growth factors undoubtedly play a role in bone formation, little is understood concerning the direct inductive/permissive capacity of bone-ECM or bone proteins themselves on human bone cells or their progenitors. Nor is the role of bone ECM in presenting growth factors understoodxe2x80x94such xe2x80x9cmatricrinexe2x80x9d (factor:ECM) interactions may be of fundamental importance in bone cell development but have not been well characterized.
The present invention provides the isolation, purification and characterization of precursors to osteoblasts, and the identification of human osteoprogenitor cells. Immunological separation of bone marrow non-adherent low-density (NALD) cells results in a marked enrichment of bone precursor cells that express osteocalcin, osteonectin, and bone alkaline phosphatase.
The bone precursor cells of the present invention, although isolatable from bone marrow, are not part of the bone marrow stromal cell compartment, nor are they a component of the hematopoietic cell lineages. The lack of a stromal cell nature is demonstrated by the failure to isolate these cells from human stromal cell isolates, and physical cell separation by density centrifugation. These cells are not hematopoietic as demonstrated by their failure to express the pan-hematopoietic cell antigen CD34, and their failure to respond to hematopoietic growth factors.
In addition to other distinguishing features, the present invention is distinct from the prior art in that the prior art studies are generally confined to osteogenic cultures in which bone cells are observed in bone marrow-derived stromal cell populations (Gronthos et al., 1994; Friedenstein et al., 1987; Luria et al., 1987; Turksen and Aubin, 1991; Van Vasselaer et al., 1994). Given the combined physical and immunological separation disclosed herein, the present population of bone precursor cells likely represents an earlier stage of bone precursors than the prior art, in that the present immune-isolated cells are not intimately associated with the endosteal surface of the bone marrow trabeculae.
Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone protein, antigen-positive cells are preosteoblasts, approximately the size of a lymphocyte, whereas other, antibody-separated subpopulations consist of osteoblasts and osteoprogenitor cells. In serum-free cultures, TGF-xcex2 stimulates the small, antigen-positive cells to become osteoblasts as these cells both increase in size, cellular complexity, and express increased levels of osteocalcin and alkaline phosphatase.
Antibody-separated cells also contain a separate population of progenitor cells that form colonies of osteoblast cells when cultured in serum-free, semi-solid media. Two types of these osteoprogenitor cells are observed: a colony-forming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells.
Osteopoietic colony-forming cells and cluster-forming cells have an obligate, but differential requirement for osteogenic growth factors. The CFCs respond to TGF-xcex2, basic fibroblast growth factor (bFGF), bone morphogenetic protein-2 (BMP-2), and 1,25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominately by 1,25-OH D3 and TGF-xcex2, but fail to respond to bFGF.
The inventors thus defined that human bone marrow contains a non-hematogenous, heterogeneous population of bone precursor cells among which exists a population of proliferating osteoprogenitor cells. The present provision of these bone precursor cell populations in sufficient numbers allows evaluation of their role in osteogenesis in both health and disease.
In one aspect, the present invention provides a process for preparing an enriched population of bone precursor cells. The process generally comprises the steps of:
(a) obtaining a population of cells that include bone precursor cells;
(b) enriching the population for bone precursor cells by exposing the population of cells to a bone precursor cell antibody immunoreactive with a bone precursor cell antigen; and
(c) removing cells of the population that do not immunoreact with a bone precursor cells antibody.
The population of cells that includes bone precursor cells may be a population of bone marrow cells, a population of cells isolated from bone, or a population of peripheral blood cells.
Bone precursor cells can be further enriched by equilibrium-density centrifugation of the population of bone marrow or peripheral blood cells. Equilibrium-density centrifugation of the cell population provides low density bone marrow cells enriched in bone precursor cells with a density of between about 1.050 and about 1.090 gm/cm3, preferably between 1.060 and 1.085 gm/cm3.
In another aspect, stromal cells present, e.g., in bone marrow cells, can be removed by exposing bone marrow cells to an adherent surface, typically tissue culture plastic or tissue culture glass.
In yet another aspect, an enriched population of bone precursor cells is further fractionated according to size. In one embodiment, size fractionation can be accomplished by fluorescence activated flow cytometry, velocity sedimentation, or counter-flow centrifugal elutriation. Bone precursor cells of the present invention generally have average diameters of between about 8 microns and about 70 microns, and preferably, of between about 10 microns and about 20 microns.
Antibodies are used to enrich the population of bone precursor cells. Suitable antibodies include any antibody immunoreactive with a bone precursor cell. Bone precursor cell antibodies particularly contemplated by the present invention include anti-osteocalcin, anti-osteonectin, and anti-bone alkaline phosphatase.
Physico-chemical separation techniques, such as equilibrium density centrifugation, can be used to obtain a moderate enrichment of bone precursor cells, e.g., to a level of about 6-7% purity. Density separation and plastic adherence are used to further increase the purity of such cells.
A significant contribution of the present invention is the use of immunoseparation techniques to obtaining substantially purified populations. The use of immuneadherence separation generates substantially pure populations of human bone precursor cells. As used herein, the term xe2x80x9csubstantially purexe2x80x9d refers to a population of bone precursor cells that is between about 60% and about 80% pure. Immuno-magnetic separation, preferably using anti-osteonectin and anti-osteocalcin antibodies, yields an almost homogeneous or essentially pure population of bone precursor cells. The term xe2x80x9cessentially purexe2x80x9d, as used herein, refers to a population of bone precursor cells that is about 95% pure.
In using a second antibody immunoreactive with a bone precursor cell antibody, enhanced enrichment of the population of bone precursor cells is thus achieved. In one embodiment, antibodies are conjugated to a solid substrate including: tissue culture plastic, agarose, other plastics, polyacrylamide, or magnetic particles.
The present invention thus provides a population of bone precursor cells enriched about 100-fold or more over the starting materials, i.e., over the bone marrow cells or peripheral blood cells that include bone precursor cells. More preferably, the population of bone precursor cells is enriched between about 1,000-fold and about 2,000-fold, or between about 2,000-fold and about 3,000-fold, or between about 3,000-fold and about 4,000-fold, over the starting cell population, with enrichment of up to about 4,800-fold being achievable.
In one embodiment, mammalian bone precursor cells are contemplated by the present invention. In a preferred embodiment, bone precursor cells from human bone marrow cells are contemplated.
The present invention further provides a composition comprising bone precursor cells. Bone precursor cells as provided herein generally have the following characteristics:
(a) immunoreactive with a bone precursor cell antibody;
(b) average cell diameter of 8 microns to about 70 microns; and
(c) differentiate into osteoblasts upon exposure to tissue growth factor xcex2, 1,25-OH Vitamin D3, basic fibroblast growth factor, or bone morphogenic protein 2.
In one aspect, the composition comprising bone precursor cells can be prepared as described above from mammalian bone, bone marrow, or peripheral blood cells. Bone precursor cells of the present invention include cells immunoreactive with anti-osteocalcin, anti-osteonectin or anti-bone alkaline phosphatase. In one embodiment, bone precursor cells express osteocalcin, osteonectin or alkaline phosphatase but do not express the pan-hematopoietic antigen CD34. In a preferred embodiment, bone precursor cells include osteoprogenitor cells and preosteoblasts.
In yet another aspect, a method of differentiating a bone precursor cell into an osteoblast is provided by the present invention. The method generally comprises the steps of:
(a) obtaining a population of bone precursor cells according to the procedure described above;
(b) exposing the bone precursor cell to a growth factor; and
(c) cultivating the bone precursor cell under serum free conditions to differentiate the bone precursor cell into an osteoblast.
Growth factors contemplated include transforming growth factor xcex2, insulin-like growth factor (IGF) and platelet-derived growth factor (AA, A/B, and B/B isoforms) 1,25-OH Vitamin D3, basic fibroblast growth factor, or bone morphogenic protein. In one embodiment, a bone precursor cell is exposed to a single growth factor. Alternatively, a bone precursor cell can be exposed to two or more growth factors.
In another embodiment, the method of differentiating a bone precursor cell into an osteoblast further comprises cultivating the bone precursor cell in the presence type I collagen, fibrinogen, fibrin, vitronectin, thrombospondin, osteocalcin, or osteonectin. In one embodiment, bone precursor cells are cultivated in the presence of type I collagen, fibrinogen and fibrin. In an alternative embodiment, bone precursor cells are cultivated in the presence of type I collagen, fibrinogen, fibrin, vitronectin, thrombospondin, osteocalcin, and osteonectin.
The present invention further provides diagnostic and prognostic methods. In certain embodiments, the invention therefore includes methods for identifying a subject at risk of developing an age-related bone disorder, which methods generally comprise the steps of:
(a) obtaining a population of cells from the subject, the population being enriched for human bone precursor cells; and
(b) quantifying the amount of a bone precursor related protein, such as, e.g., osteocalcin or osteonectin, expressed by the bone precursor cells, wherein an increased or otherwise altered amount of the protein, in comparison to the amount within the bone precursor cells of a young or middle-aged subject, is indicative of a subject at risk of developing an age-related bone disorder, such as osteoporosis.
A currently preferred example of this method includes the steps of:
(a) obtaining a population of cells from the subject, the population being enriched for human bone precursor cells; and
(b) quantifying the amount of osteocalcin or osteonectin expressed by the bone precursor cells, wherein an increased amount of osteocalcin or osteonectin, in comparison to the amount within the bone precursor cells of a young or middle-aged subject, is indicative of a subject at risk of developing an age-related bone disorder, such as osteoporosis.
These methods are generally based on the finding that osteonectin and osteocalcin antigenic expression by human preosteoblast cells increases with increasing age in a statistically significant manner. Osteonectin expression is particularly elevated in older subjects (an increase from 59 to 89 arbitrary log units).
Further methods of the invention include the diagnosis of particular groups or sub-sets of elderly subjects that have, or are at risk of developing, a certain type of bone disease or disorder, particularly osteoporosis or osteopenias or another of the group of bone disorders connected with increased aging. These methods generally comprise:
(a) obtaining a population of cells from the elderly-subject, the population being enriched for human bone precursor cells; and
(b) quantifying the amount of osteocalcin or osteonectin expressed by the bone precursor cells, wherein a decreased amount of osteocalcin or osteonectin, in comparison to the average amount within the bone precursor cells of an elderly subject, is indicative of an elderly subject having a particular type of osteoporosis, osteopenia or age-related change in bone formation.
Decreased amounts of osteocalcin and osteonectin, and most particularly, decreased amounts of osteonectin, in elderly subjects have been discovered to be indicative of an elderly subject having a particular type of osteoporosis, osteopenia or other disorder associated with age-related changes in bone formation, such as those individuals having a more severe form of osteoporosis.
This is based upon the inventors"" findings that the majority of bone precursor cells from a certain sub-set of elderly subjects belonged to an antigen-dull population. As elderly subjects with bone disorders can generally be characterized into two main groups, and as the methods of the invention generally allow two main types of bone precursor cells to be identified (one of which is the antigen-dull population), the diagnostic utility of the invention in distinguishing between these two groups is evident.
In any of the diagnostic or prognostic methods, the composition comprising the bone precursor cells will generally be prepared as described above and may be obtained from a human bone marrow or peripheral blood sample. The enrichment steps of the cell preparation method will preferably provide for a significantly purified human bone precursor cell population, will more preferably include an immunomagnetic separation step, and will most preferably include immunomagnetic separation using anti-osteocalcin and/or anti-osteonectin antibodies.
The most preferred method of quantifying the amount of osteocalcin or osteonectin expression is to use fluorescence activated flow cytometry. However, the use of other immunological methods, such as RIAs, ELISAs, and the like, is certainly contemplated; as is the use of molecular biological methods based upon the hybridization of DNA segments, probes or primers comprising osteocalcin or osteonectin sequences.